The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over Kathleen Deiteren a, ⁎ , Georgiana Surpateanu b , Kambiz Gilany c , Johan L. Willemse a , Dirk F. Hendriks a , Koen Augustyns b , Yves Laroche d , Simon Scharpé a , Anne-Marie Lambeir a a Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium b Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium c Laboratory of Protein Chemistry, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium d Department of Molecular and Cellular Medicine, University of Leuven, D. Collen Research Foundation VZW, O. and N., Herestraat 49B913, B-3000 Leuven, Belgium Received 4 August 2006; received in revised form 29 November 2006; accepted 30 November 2006 Available online 8 December 2006 Abstract The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K m and k cat , of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa–Arg substrates. CPM had the highest catalytic efficiency (k cat /K m ) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k cat and K m of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over. © 2006 Elsevier B.V. All rights reserved. Keywords: Carboxypeptidase; Structure function relationship; Membrane protein; Substrate specificity; Molecular modelling; Peptide 1. Introduction Carboxypeptidase M (CPM) belongs to the family of metallo- carboxypeptidases (metallo-CPs). This class of enzymes cata- lyzes the hydrolysis of the C-terminal peptide bond in peptides and proteins. The functions assigned to the metallo-CPs in the human body range from digestion and absorption of dietary proteins, the processing of secreted peptides and proteins and the regulation of peptide hormone activity to the modulation of protein–protein interactions [1]. Barrett et al. [2], using hierarchical, structure-based schemes for the classification of peptidases, assigned the metallo-CPs into clan MC, family M14, subfamilies M14A and M14B (also known as the CPA/CPB and CPN/CPE subfamilies respectively) [2]. More than 40% overall amino acid sequence identity is found within each subfamily, but only 15–20% between members of different subfamilies. How- ever, the residues involved in the coordination of the active site zinc ion and the key residues essential for enzyme catalysis are highly conserved for all metallo-CPs [3]. Besides CPN and CPE, the CPN/E subfamily includes the active human CPs CPM, CPD (with three CP domains, of which domain 3 does not exhibit proteolytic activity) and CPZ. Also belonging to this subfamily are a group of recently discovered homologous human proteins comprising CPX-1 and AEBP-1 [2,4]. The active members of the CPN/E subfamily exhibit a strict specificity for removing C-terminal basic amino acids Arg and Lys from peptides/ Biochimica et Biophysica Acta 1774 (2007) 267 – 277 www.elsevier.com/locate/bbapap Abbreviations: Bz, benzoyl; CPMwt, CPM wild type; CP(s), carboxypepti- dase(s); CV, column volumes; ds, dansyl; FA, furylacryloyl; GEMSA, 2- guanidinoethylmercaptosuccinic acid; GPI, glycosylphosphatidylinositol; hCPM, CPM purified from human prostasomes; MERGETPA, DL-2-mercapto- methyl-3-guanidoethylthiopropanoic acid; PEP, phosphoenolpyruvate; PI-PLC, phosphatidylinositol-specific phospholipase C ⁎ Corresponding author. Tel.: +32 3 820 25 53; fax: +32 3 820 27 34. E-mail address: kathleen.deiteren@ua.ac.be (K. Deiteren). 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.11.017